Detachabead

Buffy coats were collected from voluntary, non-remunerated, adult healthy blood donors (Sanquin Blood Supply, Amsterdam, the Netherlands), who provided written informed consent for the use of remainders of their donation for research as part of routine donor selection and blood collection procedures. Peripheral blood mononucleated cells (PBMCs) were isolated from buffy coats using a Lymphoprep (Axis-Shield PoC AS, Dundee, Scotland) density gradient. Afterward, CD19⁺ B-cells were separated using magnetic anti-CD19 Dynabeads and DETACHaBEAD (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions with purity >99%.

CD19⁺ B cells were isolated from PBMCs using Dynabeads CD19⁺ pan B (11143D, Invitrogen) according to the manufacturer’s instructions. 2.5 × 10⁸ cells of PBMCs were resuspended in 10 ml isolation buffer (PBS, 0.1% BSA, 2 mM EDTA). 250 μl of prewashed beads were added to PBMCs and incubated for 20 min in 4°C with gentle rotation. For positive isolation of CD19⁺ B cells, beads and supernatant were separated using magnet, and supernatant was discarded. Beads were washed three times, and beads bounded with CD19⁺ B cells were resuspended with 2.5 ml of cell culture medium (80% RPMI 1640, 20% heat-inactivated FBS, glutamine). CD19⁺ B cells were released from Dynabeads using DETACHaBEAD (Invitrogen, ca12506D) according to the manufacturer’s instruction.
B cells were infected with EBV to transform lymphocytes. 10 ml of B cells were transferred into a T75 flask. 1.5 ml of stock EBV collected from a B95-8 strain-containing marmoset cell line and 1 ml of phytohemagglutinin P (PHA-P) were added to flask and incubated in 37°C with a 5% CO₂ atmosphere incubator. Every 5–7 d, 10 ml of cell culture medium was added. Cells were let to grow in the CO₂ atmosphere incubator for 30 d until all B cells were transformed to LCLs.

Two human B-cell lines (GM12878 and Raji (GM04671) cells, both from Coriell Institute) and a monocytic cell line, U937 (ATCC CRL-1593.2), were cultured in RPMI 1640+GlutaMAX-I medium (Gibco), 1% penicillin-streptomycin (Sigma-Aldrich) and complement-inactivated 10% FBS (Gibco) at +37 °C and 5% CO₂.
Primary tonsillar B cells from two seronegative individuals of the study cohort were isolated with anti-CD19 magnetic beads from frozen total cell suspensions of mechanically homogenized preparations and released with DETACHaBEAD as instructed by manufacturer (Invitrogen).

Colitis was induced by AdTr of CD4⁺CD25⁻ T cells (AdTr-colitis) from spleen of MHC-compatible BALB/c mice to C.B-17 SCID recipients as previously described [9 (link)]. Briefly, splenocytes of BALB/c donor mice were subjected for positive selection of CD4⁺ T cells using Dynabeads and DETACHaBEAD (Life Technologies Europe, Ballerup, Denmark) and depletion of CD25⁺ from the CD4⁺ T cell suspensions using the CD25 MicroBead kit. Flow cytometry was used to analyze purity of the cells and showed that more than 98% of the CD4⁺ cells were CD25⁻ cells. Each recipient was reconstituted with 300,000 cells by intraperitoneal injection. Two or three weeks after transfer, peripheral blood from all mice was analyzed by flow cytometry for the presence of CD4⁺ T cells. Only animals with successful transplantation of cells were included in the study.

B cells were isolated from apheresis cones (NHS Blood and Transplant) by positive selection with CD19 Dynabeads (Life Technologies) followed by removal of the beads with Detachabead (Life Technologies). B cells were then infected with recombinant 2089 EBV at 100 MOI as previously described (Shannon-Lowe et al., 2005 (link)) and cells harvested at specific time points, washed, pelleted and frozen at −80 °C.